Active defense of plants, triggered upon recognition of an avirulence factor of a pathogen mediated by a resistance gene, follows the gene-for-gene model (Dangl and Jones, 2001, Nature 411, 826-833). To date, several plant resistance genes (R genes) have been cloned and based on the structure of the proteins they encode, the genes are divided into several groups (Hammond-Kosack and Jones, 1997, Annu. Rev. Plant Physiol. Plant Molec. Biol. 48, 575-607). Most R genes encode cytoplasmic NB-LRR proteins, containing a nucleotide binding site (NB) and leucine-rich repeats (LRR). This group consists of genes encoding CC-NB-LRR proteins, containing a coiled-coil domain and genes that encode proteins that have a domain similar to mammalian Toll and interleukin (IL) receptors, the so-called TIR-NB-LRR proteins (Hammond-Kosack and Jones, 1997, supra).
Using such specific resistance genes in breeding programs for durable resistance is problematic since pathogens easily circumvent recognition by mutations in their avirulence factors, thereby preventing induction of active defense (Westerink et al., 2004, Mol. Microbiol. 54, 533-545). Similarity among resistance proteins (R proteins) suggests the existence of common resistance pathways (Shirasu and Schulze-Lefert, 2000, Plant Mol. Biol. 44, 371-385). Therefore, identification of additional genes required for resistance not only provides information on how such signaling pathways function but might also enable us to identify genes that play a more general role in resistance. For example, by virus-induced gene silencing (VIGS) in Nicotiana benthamiana it was shown that SGT1 is involved in multiple defense pathways, such as N-, Rx- and Pto-mediated HR and resistance, and Cf-4- and Cf-9-mediated HR (Peart et al., 2002, Proc. Natl. Acad. Sci. USA 99, 10865-10869; Zhang et al., 2004, Plant J. 40, 213-224). SGT1 is an interactor of SKP1, which is a component of the SCF E3-ligase complex that is involved in ubiquitination of proteins, a modification which targets them for degradation (Schwechheimer and Schwager, 2004, Plant Cell Reports 23, 353-364). It is hypothesized that silencing an essential gene of this protein degradation system hampers the ubiquitination process, thereby inhibiting the degradation of negative regulators, which is required for defense activation (Azevedo et al., 2002, Science 295, 2073-2076).
In several resistance pathways MAPKs (mitogen activated protein kinases) are activated (Zhang and Klessig, 2001, Trends Plant Sci. 6, 520-527; Pedley and Martin, 2005, Curr. Opin. Plant Biol. 8, 541-547). In Cf-9-containing tobacco plants and cell cultures challenged with Avr9, NtWIPK (wound-induced protein kinase) and NtSIPK (salicylic acid-induced protein kinase) are activated (Romeis et al., 1999, Plant Cell 11, 273-287). VIGS of a NtCDPK (calcium-dependent protein kinase) in N. benthamiana inhibits the Cf-9/Avr9- and Cf-4/Avr4-dependent HR (Romeis et al., 2001, EMBO J. 20, 5556-5567) and VIGS of LeACIK1 (Avr/Cf-induced kinase 1) in tomato results in decreased C. fulvum resistance (Rowland et al., 2005, Plant Cell 17, 295-310). The activation of kinases during defense and the decreased resistance upon ‘knock-down’ of their encoding genes supports their function in defense activation.
Following a biased approach, 21 genes known to be involved in defense-related signaling were used for VIGS in tomato and it was found that nine of them are involved in Pto-mediated resistance. Among these are two genes encoding MAPKKs (LeMEK1 and LeMEK2) and two genes encoding MAPKs (LeNTF6 and LeWIPK) (Ekengren et al., 2003, Plant J 36, 905-917). In another study, over 2400 cDNAs from a normalized library of N. benthamiana cDNA were cloned in a Potato Virus X-based vector and used for VIGS in N. benthamiana. About 3% of the cDNAs affected Pto-dependent HR upon silencing. Among these a MAPKKKα was identified as a positive regulator of both resistance and disease (Del Pozo et al., 2004, EMBO J. 23, 3072-3082).
Lu et al. (2003, EMBO J. 22, 5690-5699) performed VIGS using 4992 cDNAs from a normalized N. benthamiana cDNA library cloned into a PVX vector. Of the cDNAs, 79 (1.6%) corresponded to genes required for Pto-mediated HR, whereas silencing of only six of them also impaired Pto-mediated resistance against Pseudomonas syringae. VIGS using a cDNA corresponding to HSP90 abolished not only Pto-mediated HR but also Pto-, Rx- and N-mediated resistance, indicating that HSP90 is required in multiple disease resistance pathways. The same set of cDNAs was also used for VIGS in N-transgenic N. benthamiana, after which the plants were inoculated with a GFP-tagged strain of TMV. Resistance against TMV was most significantly suppressed upon silencing using a cDNA fragment derived from a CC-NB-LRR-encoding gene, referred to as NRG1 (N requirement gene 1) (Peart et al., 2005, Curr. Biol. 15, 968-973). NRG1 was shown to be specifically required for N gene function, indicating that CC-NB-LRR proteins do not only act as resistance proteins involved in recognition of avirulence factors, but are also involved in the signaling pathway initiated by the TIR-NB-LRR protein N, which eventually leads to resistance (Peart et al., 2005, supra). Thus, although the tobacco NRG1 protein functions downstream of the plant's defense signaling cascade initiated by a resistance protein, it has the drawbacks that it is specifically involved in N-mediated resistance against tobacco mosaic virus (TMV) and is not a general cofactor of disease resistance (Rx- and Pto-mediated resistance against PVX and Pseudomonas syringae were unaffected by NRG1 silencing), whereby it may not be suitable for creating broad pathogen resistance in crops such as tomato.
Despite the increasing information about disease resistance pathways, there is still a need in identifying genes and proteins which can be used to create plants with durable, broad range disease resistance. It is an object of the invention to provide such nucleic acids, proteins and methods for creating plants, especially plants belonging to the family Solanaceae, with enhanced disease resistance.